At present, identification of human proteins whose sequence is not contained within one of the public databases presents a considerable analytical challenge. It is estimated that >90% of all human proteins currently fall into this category. Recently, large scale sequencing of small portions of human cDNAs has produced vast array of partial sequence information in the form of so-called Expressed Sequence Tags (Sets). It has been estimated that >50% of all human proteins are represented in the public EST database. Mann et al have shown that high quality MS/MS sequencing data can be used to successUly search these EST databases to identify human proteins. One of the major challenges in effective use of the EST database is to find a peptide fragment within the mixture of proteolytic fragments that has an EST associated with it. Because of the low average coverage of the DNA sequence of any given protein in the EST database, it is necessary to obtain high-quality MSIMS sequence information on a great many of the proteolytic peptides from the protein of interest - a challenging and time-consuming task. As an alternative strategy, we are testing the feasibility of carrying out 3 steps of N-terminal ladder sequencing chemistry on the proteolytic mixture of peptides from a protein of interest and to read out this large data set in one step from a single MALDI-TOF MS spectrum.